CA3079652C - Anti-vista antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-VISTA antibody or an antigen binding fragment thereof, a nucleic acid coding for the same, a vector carrying the nucleic acid, a cell transformed with the vector, a method for preparing the antibody or an antigen binding fragment thereof, a composition for prevention or treatment of autoimmune disease, comprising the same antibody, and a composition for concurrent administration in combination with a PD-1 antibody or PD-L1 antibody.

Description

ANTI-VISTA ANTIBODY AND USE THEREOF
[Technical Field]
[1] The present invention relates to an anti-VISTA
antibody or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector including the nucleic acid, a cell transformed with the vector, a method of preparing the antibody or antigen-binding fragment thereof, a composition including the same for preventing or treating an autoimmune disease, and a composition including the same for administration in combination with a PD-1 antibody or PD-Li antibody.

[2]
[Background Art]

[3] The immune system is a network system involving cells and tissues to respond to foreign pathogens. The immune system for responding to non-self antigens that are repeatedly encountered is broadly classified into a B-cell response using antibodies and a T-cell response that is mediated by cells. In addition to these, there are immune systems that can respond immediately to pathogens, and cells such as macrophages, NK cells and neutrophils are directly involved in removing antigens. As for the immune system, clonal selection or the like acts at the beginning of individual development to prevent the immune system from EDC_LA\M 2245789\1 1 Date Recu/Date Received 2020-04-20 acting on a self antigen. However, the immune system may often act on self antigens, which may cause disease.

[4] Rheumatoid arthritis is a typical autoimmune disease.
Various studies have shown that such an immune evasion mechanisms act even in tumors. The immune evasion mechanism can suppress or increase immunity to antigens by suppressing or promoting the activity of immune cells. Substances involved in this factor are called "immune checkpoints".
Recently, various attempts have been made to treat tumors by interfering with immunosuppressive functions on these tumors or by further strengthening factors involved in immune activity.

[5] Since antibodies blocking against CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4), which is an immune checkpoint, were first applied to patients with malignant melanoma in 2010 and a therapeutic effect thereof in about 20% of patients was clearly demonstrated, a new therapeutic approach for treating cancer by enhancing the anti-cancer immune response by inhibiting the action thereof using a therapeutic antibody targeting the immune checkpoint, which functions as a negative immune regulator, has emerged.

[6] Subsequently, the developed anti-PD-1 or anti-PD-Li antibody exhibited a better effect than the anti-CTLA-4 antibody, and in particular, exhibited a significant EDC_LA\M 2245789\1 2 Date Recu/Date Received 2020-04-20 reduction in severe side effects occurring in patients undergoing treatment with the CTLA-4 antibody, thus being very actively used in malignant melanoma, non-small cell lung cancer and bladder cancer.

[7] In particular, anti-cancer immunotherapy has been selected occasionally to date for patients for whom conventional cancer treatment methods such as surgery, anti-cancer drugs and radiation therapy have failed, but has increasingly come to be selected as a principal treatment modality based on the development of anti-PD-1/PD-L1 antibodies having reduced side effects, and is actively applied to clinical trials within the treatment scope expanded to various carcinomas not responding to conventional therapeutic agents, such as gastric cancer and liver cancer.

[8] PD-1-targeting therapeutic agents have a mechanism to activate immune surveillance of T-cells, and PD-Li is a ligand of PD-1 and binds to PD-L1 expressed in tumor cells to interfere with immunosuppression by I-cells and thereby provide anticancer activity. To further maximize this activity, a composition having an immunosuppressive mechanism that does not overlap PD-1/PD-L1 is required.
Combination therapy, in which these substances are used together with conventional therapeutic agents, is expected as a very useful tool in improving the treatment efficacy of EDC_LA\M 2245789\1 3 Date Recu/Date Received 2020-04-20 tumor patients.

[9] VISTA (V-domain-containing Ig Suppressor of T-cell Activation) is a recently identified immune checkpoint substance that is known to directly inhibit the activity of T-cells. VISTA is consistently expressed in hematopoietic compartments, is expressed at the highest level in myeloid family cells, and is expressed at relatively low levels in CD4+, CD8+ T cells and Foxp3+ CD4+ regulatory T cells. VISTA
is expressed in most immune cells, including T cells, but is known to be expressed at the highest level in myeloid cells.
Therefore, the molecule that suppresses VISTA has a target point different from PD-1 or PD-L1, which can be expected to enhance efficacy in combination therapy with PD-1/PD-L1 therapeutic agents.

[10] VISTA is structurally similar to the TIM family in that it has an extracellular immunoglobulin V (IgV) domain, and human and mouse VISTA amino acid sequences are found to have a homology of approximately 90% therebetween. Some experiments showed that VISTA inhibits proliferation of T-cells and the expression of cytokines based on, as a ligand, an antibody-presenting cell (APC) expressing the entire amino acid sequence of VISTA or VISTA-Fc having the extracellular domain of VISTA binding to the Fc portion of the antibody heavy chain. In addition, VISTA-specific antibodies are known EDC_LA\M 2245789\1 4 Date Recu/Date Received 2020-04-20 to increase the severity of disease in an autoimmune encephalomyelitis model, and also to increase anti-tumor immune activity (Le Mercier I, et al., 2014).

[11] It is also possible to develop a therapeutic agent for tumor suppression using the immunosuppressive activity of VISTA, but it is also possible to develop a therapeutic agent for GvHD (graft-versus-host disease) and autoimmune diseases by further increasing the immunosuppressive activity of VISTA. For example, antibodies against mouse VISTA were found to suppress GvHD induced by wild-type T-cells (Files, DB et al., 2011). This indicates that the activity of the antibody can act as an agonist or an antagonist depending on the epitope. Therefore, antibodies having two functions may be selected as antibodies against VISTA.

[12] Under this technical background, the present inventors have tried to develop anti-VISTA antibodies. As a result, the present inventors developed an anti-VISTA
antibody that has the desired binding ability to VISTA, and found that the anti-VISTA antibody can serve as a targeted anti-cancer agent or autoimmune disease therapeutic agent.
Based on this finding, the present invention has been completed.

[13]
EDC_LA\M 2245789\1 5 Date Recu/Date Received 2020-04-20 [Disclosure]
[Technical Problem]

[14] It is one object of the present invention to provide a novel antibody against VISTA or an antigen-binding fragment thereof.

[15] It is another object of the present invention to provide a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[16] It is another object of the present invention to provide a vector including the nucleic acid, a cell transformed with the vector, and a method of producing the same.

[17] It is another object of the present invention to provide a composition including the antibody or an antigen-binding fragment thereof for preventing or treating a tumor, a cancer or an autoimmune disease.

[18] It is another object of the present invention to provide a composition including the antibody or an antigen-binding fragment thereof for administration in combination with a PD-1 antibody or a PD-Li antibody.

[19]
[Technical Solution]

[20] In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of an antibody or an antigen-binding EDC_LA\M 2245789\1 6 Date Recu/Date Received 2020-04-20 fragment thereof binding to V-domain Ig suppressor of T-cell activation (VISTA) including: a heavy-chain variable region including: a heavy-chain CDR1 selected from the group consisting of SEQ ID NOS: 1, 7, 13 and 19;

[21] a heavy-chain CDR2 selected from the group consisting of SEQ ID NOS: 2, 8, 14 and 20; and

[22] a heavy-chain CDR3 selected from the group consisting of SEQ ID NOS: 3, 9, 15 and 21; and

[23] a light-chain variable region including: a light-chain CDR1 selected from the group consisting of SEQ ID NOS:
4, 10, 16 and 22;

[24] a light-chain CDR2 selected from the group consisting of SEQ ID NOS: 5, 11, 17 and 23; and

[25] a light-chain CDR3 selected from the group consisting of SEQ ID NOS: 6, 12, 18, 24, and 34 to 40.

[26] In another aspect of the present invention, provided is a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[27] In another aspect of the present invention, provided is a vector including the nucleic acid.

[28] In another aspect of the present invention, provided is a cell transformed with the vector.

[29] In another aspect of the present invention, provided is a method of producing the antibody or antigen-binding EDC_LA\M 2245789\1 7 Date Recu/Date Received 2020-04-20 fragment thereof including: (a) culturing the cell; and (b) recovering an antibody or antigen-binding fragment thereof from the cultured cell.

[30] In another aspect of the present invention, provided is a composition including the antibody or an antigen-binding fragment thereof as an active ingredient for preventing or treating a tumor, a cancer or an autoimmune disease. In another aspect of the present invention, provided is a method of preventing or treating a tumor, a cancer or an autoimmune disease including administering the antibody or antigen-binding fragment thereof to a patient with a tumor, a cancer or autoimmune disease. In another aspect of the present invention, provided are the use of the antibody or antigen-binding fragment thereof for the inhibition of the immunosuppressive mechanism of VISTA and the use of the antibody or antigen-binding fragment thereof for the prevention or treatment of a tumor, a cancer or the use of the antibody or antigen-binding fragment thereof for the improvement of the immunosuppressive ability of VISTA and the use of the antibody or antigen-binding fragment thereof for the prevention or treatment of an autoimmune disease.

[31] In another aspect of the present invention, provided is a composition including the antibody or antigen-binding fragment thereof for administration in combination with a PD-1 antibody or a PD-L1 antibody. In another aspect of the EDC_LA\M 2245789\1 8 Date Recu/Date Received 2020-04-20 present invention, provided is a method of preventing or treating a tumor or a cancer including administering the antibody or an antigen-binding fragment thereof in combination with a PD-1 antibody or a PD-L1 antibody to a patient. In another aspect of the present invention, provided are the use of the antibody or antigen-binding fragment thereof for administration in combination with a PD-1 antibody or a PD-L1 antibody to treat a tumor or a cancer.

[32]
[Advantageous Effects]

[33] The anti-VISTA antibody or antigen-binding fragment thereof according to the present invention exhibits desired binding ability to VISTA and can be useful for the prevention or treatment of a targeted cancer/tumor or autoimmune disease.
The present invention enables the development of a therapeutic agent having a target point different from that of a conventional therapeutic agent targeting an immune checkpoint, thereby providing combination therapy with the conventional therapeutic agent and single therapy for tumor treatment, and can enhance the activity of checkpoints suppressing immune activity, thereby providing a novel immunosuppressive therapy.

[34]
[Description of Drawings]

[35] FIG. 1 shows the result of determination of whether EDC_LA\M 2245789\1 9 Date Recu/Date Received 2020-04-20 or not VISTA-expressing cells bind to monoclonal scEv phages by reacting monoclonal scEv phages with CHO-K1 cells expressing VISTA.

[36] FIG. 2 shows the result of measurement of ELISA to determine whether or not cross-reactivity exists between the selected anti-VISTA antibody and human and mouse VISTA
antigens.

[37] FIG. 3 shows the result of determination of the binding ability of selected anti-VISTA antibody to CHO-K1 cells expressing human and mouse VISTA.

[38] FIG. 4 shows the result of measurement of the IFN-y produced in the human peripheral blood allo-MLR reaction by ELISA.

[39] FIG. 5 shows a result determining that an anti-VISTA
antibody administration group loses a significant amount of weight.

[40] FIG. 6 shows a result determining that the anti-VISTA antibody administration group undergoes a significant reduction in tumor weight.

[41] FIG. 7 shows a result determining the effective proliferation of cells through a mixed lymphocyte reaction (MLR) in order to evaluate the function of the optimized anti-VISTA antibody.
EDC_LA\M 2245789\1 10 Date Recu/Date Received 2020-04-20

[42] FIG. 8 shows a result determining increased cytokine secretion through a mixed lymphocyte reaction (MLR) in order to evaluate the function of the optimized anti-VISTA antibody.

[43] FIG. 9 shows a result determining that an optimized anti-VISTA antibody administration group loses a significant amount of weight.

[44] FIG. 10 shows a result determining that the optimized anti-VISTA antibody administration group undergoes a significant reduction in tumor weight.

[45]

[46]
[Best Mode]

[47] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.

[48] Thus, in one aspect, the present invention is directed to an antibody binding to V-domain Ig suppressor of T-cell activation (VISTA) or an antigen-binding fragment thereof including: a heavy-chain variable region including:
a heavy-chain CDR1 selected from the group consisting of SEQ
ID NOS: 1, 7, 13 and 19;

[49] a heavy-chain CDR2 selected from the group consisting EDC_LA\M 2245789\1 11 Date Recu/Date Received 2020-04-20 of SEQ ID NOS: 2, 8, 14 and 20; and

[50] a heavy-chain CDR3 selected from the group consisting of SEQ ID NOS: 3, 9, 15 and 21; and

[51] a light-chain variable region including: a light-chain CDR1 selected from the group consisting of SEQ ID NOS:
4, 10, 16 and 22;

[52] a light-chain CDR2 selected from the group consisting of SEQ ID NOS: 5, 11, 17 and 23; and

[53] a light-chain CDR3 selected from the group consisting of SEQ ID NOS: 6, 12, 18, 24, and 34 to 40.

[54] As used herein, the term "antibody" refers to an anti-VISTA antibody that specifically binds to VISTA. The scope of the present invention includes not only a complete antibody specifically binding to VISTA but also an antigen-binding fragment of the antibody molecule.

[55] The term "complete antibody" refers to a structure having two full-length light chains and two full-length heavy chains, wherein each light chain is linked to a corresponding heavy chain by a disulfide bond. The heavy-chain constant region has gamma (y), mu (p), alpha (a), delta (5) and epsilon (s) types, and is subclassified into gamma 1 (yl), gamma 2 (y2), gamma 3 (y3), gamma 4 (y4), alpha 1 (al) and alpha 2 (a2). The constant region of the light chain has kappa (K) and lambda (A) types.

[56] The antigen-binding fragment of an antibody or EDC_LA\M 2245789\1 12 Date Recu/Date Received 2020-04-20 antibody fragment refers to a fragment that at least has antigen-binding capacity and includes Fab, F(ab'), F(ab')2, Fv and the like. Among the antibody fragments, Fab refers to a structure including a variable region of each of the heavy chain and the light chain, the constant region of the light chain, and the first constant domain (CH1) of the heavy chain, each having one antigen-binding site. Fab is different from Fab in that it further includes a hinge region including at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 is created by a disulfide bond between cysteine residues in the hinge region of Fab'.
Fv is the minimal antibody fragment having only a heavy-chain variable region and a light-chain variable region, and recombinant technology for producing Fv is disclosed in PCT
International Publications such as WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. Two-chain Fv is a fragment wherein the variable region of the heavy chain and the variable region of the light chain are linked by a non-covalent bond, and single-chain Fv (scFv) is a fragment wherein the variable region of the heavy chain and the variable region of the light chain are generally linked by a covalent bond via a peptide linker therebetween, or are directly linked at the C-terminal, forming a dimer-shaped structure, like the two-chain Fv. Such antibody fragments may be obtained using proteases (e.g., Fab can be obtained EDC_LA\M 2245789\1 13 Date Recu/Date Received 2020-04-20 by restriction-cleaving the whole antibody with papain, and the F(ab')2 fragment can be obtained by restriction-cleaving the whole antibody with pepsin), and may be prepared using genetic recombination techniques.

[57] In one embodiment, the antibody of the present invention is in an Fv form (for example, scFv) or a complete antibody form. In addition, the heavy-chain constant region may be selected from gamma (y), mu (u), alpha (a), delta (5) and epsilon (c) isotypes. For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light-chain constant region may be kappa or lambda.

[58] As used herein, the term "heavy chain" encompasses both a full-]ength heavy chain, which includes a variable domain (VH) containing an amino acid sequence having a variable region sequence sufficient for imparting specificity to an antigen and three constant domains (CH1, CH2 and CH3), and a fragment thereof. As used herein, the term "light chain" encompasses both a full-length light chain, which includes a variable domain (VL) containing an amino acid sequence having a variable region sequence sufficient for imparting specificity to an antigen and a constant domain (CL), and a fragment thereof.

[59] The antibody of the present invention includes, but is not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric EDC_LA\M 2245789\1 14 Date Recu/Date Received 2020-04-20 antibodies, single-chain Fvs (scFVs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bond Fvs (sdFVs), anti-idiotypic (anti-Id) antibodies, epitope-binding fragments of such antibodies, and the like.

[60] The term "monoclonal antibody" refers to an identical antibody, excluding possible naturally occurring mutations where an antibody obtained from a population of substantially homogeneous antibodies, that is, each antibody constituting the population, may be present in a small amount. Monoclonal antibodies are highly specific and are thus induced against a single antigenic site. Unlike conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[61] The term "epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes usually consist of a group of chemically active surface molecules, such as amino acid or sugar side chains, and generally have not only specific three-dimensional structural characteristics but also specific charge characteristics.
Three-dimensional epitopes are distinguished from non-three-dimensional epitopes in that the bond to the former is broken in the presence of a denatured solvent, while the bond to the latter is not broken.
EDC_LA\M 2245789\1 15 Date Recu/Date Received 2020-04-20

[62] The non-human (e.g., murine) antibody of the "humanized" form is a chimeric antibody containing a minimal sequence derived from non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which a residue from the hypervariable region of a receptor is replaced with a residue from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate having the desired specificity, affinity and ability.

[63] As used herein, the term "human antibody" refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences constituting the antibody including a complementarity-determining region and a structural region are composed of human immunoglobulins.

[64] The human antibody includes not only a "chimeric"
antibody (immunoglobulin) in which the heavy-chain and/or light-chain portions are derived from a certain species, or are identical or homologous to the corresponding sequences in an antibody belonging to a certain antibody class or subclass, but the remaining chain(s) are derived from another species or are identical or homologous to the corresponding sequences in an antibody belonging to another antibody class or subclass, but also a fragment of the antibody that exhibits the desired biological activity.

[65] As used herein, the term"antibody variable domain"
EDC_LA\M 2245789\1 16 Date Recu/Date Received 2020-04-20 refers to light-chain and heavy-chain regions of an antibody molecule including the amino acid sequence of a complementarity-determining region (CDR; i.e., CDR1, CDR2, and CDR3) and a framework region (FR). VH refers to a variable domain of the heavy chain. VL refers to a variable domain of the light chain.

[66] The term "complementarity-determining region" (CDR;
i.e., CDR1, CDR2, and CDR3) refers to an amino acid residue of the antibody variable domain that is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[67] In the present invention, the antibody binding to VISTA or an antigen-binding fragment thereof may include a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5 and a light-chain CDR3 of SEQ ID NO: 6,

[68] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 33,

[69] a heavy-chain variable region including a heavy-EDC_LAM 2245789\1 17 Date Recu/Date Received 2020-04-20 chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 34,

[70] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 35,

[71] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 36,

[72] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 37,

[73] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
EDC_LA\M 2245789\1 18 Date Recu/Date Received 2020-04-20 2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 38,

[74] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 39,

[75] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO:
2 and a heavy-chain CDR3 of SEQ ID NO: 3, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
4, a light-chain CDR2 of SEQ ID NO: 5, and a light-chain CDR3 of SEQ ID NO: 40,

[76] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 7, a heavy-chain CDR2 of SEQ ID NO:
8 and a heavy-chain CDR3 of SEQ ID NO: 9, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
10, a light-chain CDR2 of SEQ ID NO: 11, and a light-chain CDR3 of SEQ ID NO: 12,

[77] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 13, a heavy-chain CDR2 of SEQ ID NO:
14 and a heavy-chain CDR3 of SEQ ID NO: 15, and a light-chain EDC_LA\M 2245789\1 19 Date Recu/Date Received 2020-04-20 variable region including a light-chain CDR1 of SEQ ID NO:
16, a light-chain CDR2 of SEQ ID NO: 17, and a light-chain CDR3 of SEQ ID NO: 18, or

[78] a heavy-chain variable region including a heavy-chain CDR1 of SEQ ID NO: 19, a heavy-chain CDR2 of SEQ ID NO:
20 and a heavy-chain CDR3 of SEQ ID NO: 21, and a light-chain variable region including a light-chain CDR1 of SEQ ID NO:
22, a light-chain CDR2 of SEQ ID NO: 23, and a light-chain CDR3 of SEQ ID NO: 24.

[79] The term "framework region" (FR) refers to a variable-domain residue other than a CDR residue. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[80] The "Fv" fragment is an antibody fragment containing complete antibody recognition and binding sites. Such a region includes a dimer, for example, scFv, that consists of one heavy-chain variable domain and one light-chain variable domain substantially tightly covalently connected to each other.

[81] A "Fab" fragment contains a variable domain and a constant domain of the light chain and a variable domain and a first constant domain (CH1) of the heavy chain. A F(ab')2 antibody fragment generally includes a pair of Fab fragments covalently linked via a hinge cysteine located therebetween near the carboxyl end thereof.
EDC_LA\M 2245789\1 20 Date Recu/Date Received 2020-04-20

[82] The "single chain Fv" or "scFv" antibody fragment includes VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain in order for the scFv to form a desired structure for antigen binding.

[83] The VISTA antibody may include single or double chains. Functionally, the binding affinity of the VISTA
antibody ranges from 10-5 M to 10-12 M. For example, the binding affinity of the VISTA antibody is 10-6 M to 10-12 M, 10-7 M to 10-12 M, 10-8 M to 10-12 M, 10-9 M to 10-12 M, 10-5 M to 10-11 M, 10-6 M to 10-11 M, 10-7 M to 10-11 M, 10-8 M to 10-11 M, 10-9 M to 10-11 M, 10-10 M to 10-11 M, 10-5 M to 10-1 M, 10-6 M
to 10' m, 10-7 M to 10-10 M, 10-8 M to 10-1 M, 10-9 M to 10-n m, 10-5 M to 10-9 M, 10-6 M to 10-9 M, 10-7 M to 10-9 M, 10-8 M to 10-9 M, 10-5 M to 10-8 Mf 10-6 M to 10-8M, 10-7 M to 10-M, 10-5 M to 10-7 M, 10-6 M to 10-7 M, or 10-5 M to 10-6 M.

[84] The antibody binding to VISTA or an antigen-binding fragment thereof may include a heavy-chain variable region selected from the group consisting of SEQ ID NOS: 25, 27, 29, 31 and 49. In addition, the antibody binding to VISTA or an antigen-binding fragment thereof may include a light-chain variable region selected from the group consisting of SEQ ID
NOS: 26, 28, 30, 32, 41 to 48, and 50.

[85] In a specific embodiment according to the present EDC_LA\M 2245789\1 21 Date Recu/Date Received 2020-04-20 invention, the antibody binding to VISTA or the antigen-binding fragment thereof may include: the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 26;

[86] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 41;

[87] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 42;

[88] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 43;

[89] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 44;

[90] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 45;

[91] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 46;

[92] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 47;

[93] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 48;

[94] the heavy-chain variable region of SEQ ID NO: 25 and the light-chain variable region of SEQ ID NO: 50;

[95] the heavy-chain variable region of SEQ ID NO: 49 and the light-chain variable region of SEQ ID NO: 48;

[96] the heavy-chain variable region of SEQ ID NO: 49 and EDC_LA\M 2245789\1 22 Date Recu/Date Received 2020-04-20 the light-chain variable region of SEQ ID NO: 50;

[97] the heavy-chain variable region of SEQ ID NO: 27 and the light-chain variable region of SEQ ID NO: 28;

[98] the heavy-chain variable region of SEQ ID NO: 29 and the light-chain variable region of SEQ ID NO: 30; or

[99] the heavy-chain variable region of SEQ ID NO: 31 and the light-chain variable region of SEQ ID NO: 32.

[100] "Phage display" is a technique for displaying a mutant polypeptide as a fusion protein with at least a part of a coat protein on the surface of the particle of a phage, for example a fibrous phage. The usefulness of phage display is to rapidly and efficiently classify sequences that bind to target antigens with high affinity in large libraries of randomized protein mutants. Displaying peptides and protein libraries on phages has been used to screen millions of polypeptides in order to identify polypeptides with specific binding properties.

[101] Phage display technology has offered a powerful tool for producing and screening novel proteins that bind to specific ligands (e.g., antigens). Using phage display technology, large libraries of protein mutants can be generated and sequences binding with high affinity to target antigens can be rapidly classified. The nucleic acid encoding mutant polypeptides is fused with a nucleic acid sequence encoding a viral coat protein, e.g., a gene III or gene VIII
EDC_LA\M 2245789\1 23 Date Recu/Date Received 2020-04-20 protein. A monophasic phage display system, in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a part of the gene III protein, has been developed. In the monophasic display system, a fused gene is expressed at a low level, a wild-type gene III protein is also expressed, and thus particle infectivity is maintained.

[102] It is important to demonstrate the expression of peptides on the fibrous phage surface and the expression of functional antibody fragments in the peripheral cytoplasm of E. coli for the development of antibody phage display libraries. Libraries of antibody- or antigen-binding polypeptides are produced through a number of methods, for example, methods of modifying a single gene by inserting a random DNA sequence or cloning a related gene sequence.
Screening can be performed in the libraries regarding the expression of antibody- or antigen-binding proteins having desired characteristics.

[103] Phage display technology has several advantages over conventional hybridomas and recombinant methods for producing antibodies having desired characteristics. This technique provides the generation of large antibody libraries with a variety of sequences within a short time without using animals. The production of hybridomas and the production of humanized antibodies may require a production time of several EDC_LA\M 2245789\1 24 Date Recu/Date Received 2020-04-20 months. In addition, since no immunity is required, the phage antibody libraries can generate antibodies against antigens that are toxic or have low antigenicity. The phage antibody libraries can also be used to produce and identify novel therapeutic antibodies.

[104] Techniques for generating human antibodies from immunized or non-immunized human, germline sequences, or naive B cell Ig repertoires using phage display libraries can be used. Naive or non-immunogenic antigen-binding libraries can be produced using various lymphatic tissues.

[105] Techniques for identifying and separating high-affinity antibodies from phage display libraries are important for the separation of new therapeutic antibodies.
The separation of high-affinity antibodies from the libraries depends on the size of the libraries, the production efficiency in bacterial cells and the variety of libraries.
The size of the libraries is reduced by improper folding of the antibody- or antigen-binding protein and inefficient production due to the presence of the stop codon. Expression in bacterial cells may be inhibited by improper folding of the antibody- or antigen-binding domain. The expression can be improved by alternately mutating residues on the surface of the variable/constant interfaces or the selected CDR
residues. The sequence of the framework region is an element that enables proper folding when generating antibody phage EDC_LA\M 2245789\1 25 Date Recu/Date Received 2020-04-20 libraries in bacterial cells.

[106] It is important to generate various libraries of antibody- or antigen-binding proteins in the separation of high-affinity antibodies. CDR3 regions have often been found to participate in antigen binding. Since the CDR3 region on the heavy chain varies considerably in terms of size, sequence and structural/dimensional morphology, various libraries can be produced using the same.

[107] In a specific embodiment according to the present invention, antibody optimization was performed to enhance the affinity of the anti-VISTA antibody clone 1B8, and random mutations were introduced into the light-chain CDR3 and heavy-chain CDR3 of 1B8 and biopanning was performed using a mutant scFv phage library. As a result, 8 types of optimized clones including at least one light-chain variable region CDR3 selected from the group consisting of SEQ ID NOS: 33 to 40 were obtained.

[108] Also, diversity can be created by randomizing the CDR regions of variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids results in production of antibody sequences having increased diversity and an increased chance of identifying new antibodies.

[109] The antibody or antibody fragment of the present invention may include the sequence of the antibody mentioned EDC_LA\M 2245789\1 26 Date Recu/Date Received 2020-04-20 herein as well as biological equivalents thereto, as long as it can specifically recognize VISTA. For example, additional variations can be made to the amino acid sequence of the antibody in order to further improve the binding affinity and/or other biological properties of the antibody. Such variations include, for example, deletion, insertion and/or substitution of the amino acid sequence residues of the antibody. Such amino acid variations are based on the relative similarity of amino acid side chain substituents, such as the hydrophobicity, hydrophilicity, charge and size thereof. It can be seen through analysis of the size, shape and type of amino acid side chain substituents that all of arginine, lysine and histidine are positively charged residues; alanine, glycine and serine have similar sizes;
and phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are considered to be biologically functional equivalents.

[110] When taking into consideration variations having biologically equivalent activity, the antibody or a nucleotide molecule encoding the same according to the present invention is interpreted to include a sequence having substantial identity with the sequence set forth in the sequence number. The term "substantial identity" means EDC_LA\M 2245789\1 27 Date Recu/Date Received 2020-04-20 that a sequence has a homology of at least 90%, most preferably a homology of at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, when aligning the sequence of the present invention and any other sequence so as to correspond to each other as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. Alignment methods for sequence comparison are well-known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible through NCBI or the like, and can be used in conjunction with sequence analysis programs such as BLASTP, BLASTM, BLASTX, TBLASTN and TBLASTX
over the Internet.

[111] Based on this, the antibody or antigen-binding fragment thereof according to the present invention can have a homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to the sequence disclosed herein or the entirety thereof. Such homology can be determined through sequence comparison and/or alignment by methods known in the art. For example, the percentage sequence homology of the nucleic acid or protein according to the present invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

Date Recue/Date Received 2021-06-29

[112] In another aspect, the present invention is directed to a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[113] By isolating the nucleic acid encoding the antibody or antigen-binding fragment thereof according to the present invention, an antibody or antigen-binding fragment thereof can be produced in a recombinant manner. The nucleic acid is isolated and inserted into a replicable vector, followed by further cloning (amplification of DNA) or further expression. Based on this, in another aspect, the present invention is directed to a vector including the nucleic acid.

[114] The term "nucleic acid" is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and a nucleotide, which is a basic constituent unit of a nucleic acid, includes naturally derived nucleotides as well as analogues thereof, wherein sugar or base moieties are modified. The sequence of the nucleic acid encoding heavy- and light-chain variable regions of the present invention can vary. Such variation includes addition, deletion, or non-conservative or conservative substitution of nucleotides.

[115] The DNA encoding the antibody can be easily separated or synthesized using conventional procedures (for example, using an oligonucleotide probe capable of specifically binding to DNA encoding heavy and light chains of the EDC_LA\M 2245789\1 29 Date Recu/Date Received 2020-04-20 antibody). A variety of vectors are obtainable. Vector components generally include, but are not limited to, one or more of the following components: signal sequences, replication origins, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[116] As used herein, the term "vector" refers to a means for expressing target genes in host cells, and includes plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

[117] The term "operably linked" means a functional linkage between a nucleic acid expression regulation sequence (e.g., array of the binding site of promoter, signal sequence or transcription regulator) and another nucleic acid sequence, and enables the regulation sequence to regulate the transcription and/or translation of the other nucleic acid sequence.

[118] When a prokaryotic cell is used as a host, it generally includes a potent promoter capable of conducting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, 1pp promoter, pLk promoter, pR)\, promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosome-binding site for EDC_LA\M 2245789\1 30 Date Recu/Date Received 2020-04-20 initiation of translation and a transcription/translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, it includes a promoter derived from the genome of mammalian cells (e.g., a metallothionein promoter, a P-actin promoter, a human hemoglobin promoter or a human muscle creatine promoter), or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and generally has a polyadenylation sequence as a transcription termination sequence.

[119] Optionally, the vector may be fused with another sequence in order to facilitate purification of the antibody expressed therefrom. The sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6x His (hexahistidine; Qiagen, USA) and the like.

[120] The vector includes antibiotic-resistant genes commonly used in the art as selectable markers, and examples thereof include genes conferring resistance to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.
EDC_LA\M 2245789\1 31 Date Recu/Date Received 2020-04-20

[121] In another aspect, the present invention is directed to a cell transformed with the above-mentioned vector. The cell used to produce the antibody of the present invention may be a prokaryote, yeast or higher eukaryotic cell, but is not limited thereto.

[122] Prokaryotic host cells such as Escherichia coli, strains of the genus Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces spp., Pseudomonas spp.
(for example, Pseudomonas putida), Proteus mirabilis and Staphylococcus spp. (for example, Staphylococcus carnosus) may be used.

[123] Interest in animal cells is the greatest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CH0/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, and HT1080.

[124] In another aspect, the present invention is directed to a method of producing the antibody or antigen-binding fragment thereof including: (a) culturing the cell; and (b) recovering an antibody or antigen-binding fragment thereof from the cultured cell.

[125] The cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other essential supplements well-EDC_LAW\ 2245789\1 32 Date Recu/Date Received 2020-04-20 known to those skilled in the art may be included in appropriate concentrations. Culture conditions such as temperature and pH are conventionally used with host cells selected for expression, as will be apparent to those skilled in the art.

[126] The recovery of the antibody or antigen-binding fragment thereof can be carried out, for example, by centrifugation or ultrafiltration to remove impurities and purification of the resulting product using, for example, affinity chromatography. Other additional purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite (HA) chromatography may be used.

[127] In another aspect, the present invention is directed to a composition for preventing or treating a tumor or a cancer including the antibody as an active ingredient. The antibody may be IgG or a fragment including a variable region, namely ScFv or Fab. In addition, the variable region of the heavy chain may be IgGl, IgG2, IgG3 or IgG4.

[128] The present invention may provide, for example, a pharmaceutical composition for preventing or treating a tumor or a cancer containing: (a) a pharmaceutically effective amount of the antibody to VISTA or antigen-binding fragment thereof according to the present invention; and (b) a pharmaceutically acceptable carrier. The present invention EDC_LA\M 2245789\1 33 Date Recu/Date Received 2020-04-20 also relates to a method for preventing or treating a tumor or a cancer including administering the antibody or an antigen-binding fragment thereof according to the present invention to a patient with a tumor or a cancer. The present invention may provide the use of the antibody or antigen-binding fragment thereof for the inhibition of the immunosuppressive mechanism of VISTA and the use thereof for the prevention or treatment of a tumor or a cancer.

[129] Tumors, which are the diseases to which the composition can be applied, include typical tumors or cancers that respond to immunotherapy, as well as tumors or cancers that have not been treated with immunotherapy to date. Non-limiting examples of tumors or cancers that are targets of treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic carcinomas. In addition, the tumors or cancers according to the present invention include refractory or recurrent cancers that can be treated using the antibody of the present invention.
EDC_LA\M 2245789\1 34 Date Recu/Date Received 2020-04-20

[130] In another aspect, the present invention is directed to a composition for preventing or treating an autoimmune disease including the antibody as an active ingredient. The antibody may be IgG or a fragment including a variable region, namely ScFv or Fab. In addition, the variable region of the heavy chain may be IgGl, IgG2, IgG3 or IgG4.

[131] The present invention may provide, for example, a composition for preventing or treating an autoimmune disease, containing: (a) a pharmaceutically effective amount of the antibody to VISTA or antigen-binding fragment thereof according to the present invention; and (b) a pharmaceutically acceptable carrier. The present invention also relates to a method for preventing or treating an autoimmune disease including administering the antibody or an antigen-binding fragment thereof according to the present invention to a patient with an autoimmune disease. The present invention may provide the use of the antibody or antigen-binding fragment thereof for the improvement of the immunosuppressive mechanism of VISTA and the use thereof for the prevention or treatment of an autoimmune disease.

[132] The autoimmune disease may for example include:
leukemia; chronic fatigue syndrome; graft-versus-host disease (GVHD); hyperalgesia; inflammatory bowel diseases;
neuroinflammatory diseases; ischemia/ reperfusion injuries including cerebral ischemia, brain damage resulting from EDC_LA\M 2245789\1 35 Date Recu/Date Received 2020-04-20 trauma, epilepsy, bleeding or seizures that may cause neurodegeneration; diabetes, for example, type 1 combustible diabetes; multiple sclerosis; eye diseases; pain;
pancreatitis; pulmonary fibrosis; rheumatoid diseases such as rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis, seropositive polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis, Sjogren's syndrome, rheumatic fever, polychondritis and multiple myalgia, rheumatoid and giant cell arteritis; septic shock; side effects resulting from radiotherapy; systemic lupus erythematosus; temporal mandibular joint disease; thyroiditis and the like.

[133] As used herein, the term "prevention" refers to any action causing the suppression of a cancer or an autoimmune disease or the delay of progression thereof by administration of the composition according to the present invention. The term "treatment" means suppression, alleviation or elimination of the progression of cancer, or suppression, alleviation or elimination of the progress of an autoimmune disease.

[134] In some cases, it is possible to effectively target tumor cells using the antibody in combination with another anti-cancer therapeutic agent, and it is possible to enhance EDC_LA\M 2245789\1 36 Date Recu/Date Received 2020-04-20 the immune response targeting tumor cells by increasing anti-tumor T cell activity. The antibody may be used in combination with: other anti-neoplastic or immunogenic agents [e.g., attenuated cancer cells, tumor antigens (including recombination proteins, peptides and carbohydrate molecules), antigen transfer cells, for example, tumor-derived antigens or nucleic acids-pulsed dendritic cells, immunostimulating cytokines (e.g., IL-2, IFNa2, and GM-CSF), and cells transfected with genes encoding immunostimulating cytokines (including for example but not limited to GM-CSF)];
standard cancer therapy (e.g. chemotherapy, radiotherapy or surgery); or other antibodies (including but not limited to VEGF, EGFR, Her2/neu, VEGF receptors, other growth factor receptors, CD20, CD40, CTLA-4, OX-40, 4-IBB, and ICOS).

[135] In another aspect, the present invention is directed to a composition containing the antibody or an antigen-binding fragment thereof for administration in combination with a PD-1 antibody or a PD-Li antibody. In another aspect of the present invention, provided is a method of preventing or treating a tumor or a cancer including administering the antibody or an antigen-binding fragment thereof in combination with a PD-1 antibody or a PD-Li antibody to a patient with a tumor or a cancer. In another aspect of the present invention, provided are the use of the antibody or antigen-binding fragment thereof for administration in EDC_LA\M 2245789\1 37 Date Recu/Date Received 2020-04-20 combination with a PD-1 antibody or a PD-Li antibody to treat a tumor or a cancer.

[136] The antibody or antigen-binding fragment thereof may be administered simultaneously with a PD-1 antibody or a PD-Li antibody, or may be administered separately therefrom with a time interval therebetween. The separate administration of the PD-1 antibody or PD-Li antibody may be performed before or after administration of the antibody or antigen-binding fragment thereof.

[137] Since the composition uses the anti-VISTA antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient, descriptions in common therebetween are omitted.

[138] The pharmaceutically acceptable carrier contained in the pharmaceutical composition may include a pharmaceutically acceptable carrier commonly used in preparations, and may include, but without being limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The composition according to the present invention may further contain a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspension EDC_LA\M 2245789\1 38 Date Recu/Date Received 2020-04-20 agent, a preservative, or the like, in addition to the ingredients described above.

[139] The pharmaceutical composition according to the present invention may be administered orally or parenterally.
The parenteral administration may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, rectal administration or the like.

[140] Upon oral administration, since proteins or peptides are digested, an oral composition should be coated with an active drug or formulated so as to protect the same from degradation in the stomach. In addition, the pharmaceutical composition may be administered using any device capable of delivering the active substance to target cells.

[141] The suitable dose of the pharmaceutical composition according to the present invention may vary depending on factors such as the formulation method, administration method, and age, body weight, gender, pathological conditions, diet, administration time, administration route, excretion rate and responsiveness of the patient, and a general physician of ordinary skill can easily determine and prescribe a dose effective for the desired treatment or prevention. For example, the daily dose of the pharmaceutical composition according to the present EDC_LA\M 2245789\1 39 Date Recu/Date Received 2020-04-20 invention may be within the range of 0.001 to 100 mg/kg.
The term "pharmaceutically effective amount" may mean an amount sufficient to prevent or treat a cancer or autoimmune disease.

[142] The pharmaceutical composition according to the present invention may be prepared into a unit dose form, or may be incorporated into a multi-dose container through formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily implemented by those skilled in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, a suspension, a syrup or an emulsion in an oil or aqueous medium, or may be in the form of an extract, a powder, a granule, a tablet or a capsule.
The composition may further contain a dispersant or a stabilizer.

[143]

[144] Example

[145] Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.

[146]
EDC_LA\M 2245789\1 40 Date Recu/Date Received 2020-04-20

[147] Example 1. Screening of antibodies binding to VISTA

[148] Human naive ScFv (scFv) libraries disclosed in Korean Patent Laid-Open No. 10-2008-0109417 were used for antibody libraries for screening antibodies binding to VISTA and preparation of the libraries. 100 pl of an antigen (hVISTA-Fc, R&D systems. Cat. No. 7126-B7) was plated at 2 p1/ml onto a 96-well immunoplate and incubated for 4 C overnight. The next day, the antigen-coated plate was washed three times with PBS, and was then reacted with 200 pl of 2% BSA blocking buffer at room temperature for 2 hours. 50 pl of XL1-Blue stock was added to 2 ml of 2x YT-TET (tetracycline 10 pg/ml) growth medium and incubated at 37 C for 2 hours at 200 rpm, and 13 ml thereof was further added and incubated until OD600 reached 0.5. After blocking for 2 hours, the well was washed 3 times with 1XPBS. The phage library group was combined with each washed well, and the phage library and 4% BSA were mixed in equal amounts, and then 200 pl of the mixture was added thereto, followed by rocking at room temperature for 30 minutes and allowing a reaction to proceed for 2 hours. After the phage library reaction was completed, the supernatant was discarded, the residue was washed 5 times with 0.05% PBST
and washed 5 times with PBS, 100 pl of 100 mM TEA
(trimethylamine) was added to each well, and the resulting mixture was shaken at room temperature for 10 minutes. After minutes, 50 pl of 1M Tris (pH 7.5) was added to each well, EDC_LA\M 2245789\1 41 Date Recu/Date Received 2020-04-20 followed by mixing. The supernatant was added to 10 ml of XL1-blue having OD600 of 0.5 to conduct infection at 37 C for 30 minutes. After the infection was completed, 100 pl was used as an output titer and the residue was centrifuged at 7,000 rpm for 10 minutes. The supernatant was discarded and the precipitate was spread over a large square plate (CM 34 ug/m1 + 1% glucose) and incubated at 30 C overnight. The 100 pl left as an output titer was diluted to 1/10, 1/100, and 1/1000, spread on a CM plate and incubated overnight at 37 C.
The next day, colonies grown on the square plate were scraped using a loop after the addition of 50 ml of 2x YT medium thereto and centrifuged at 7,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was used to prepare a primary panning stock. 100 ml of a 2x YT culture medium (growth medium: CM 34 pg/ml + 1% glucose) was placed in a 500 ml Erlenmeyer flask, cells were added thereto to adjust the 0D600 to 0.2 and were grown at 200 rpm and 37 C to adjust the OD600 to 0.5. After the cells were cultured until the OD600 reached 0.5, a helper phage (M13K07 mutant) was added in an amount 20 times that of the cells. After infection with the helper phage at 37 C for 30 minutes, centrifugation was performed at 7,000 rpm for 10 minutes. The supernatant was discarded and the cells were incubated overnight in 100 ml of a fresh 2xYT medium (CM 34 ug/m1 + Kan. 70 pg/ ml + 1 mM IPTG + 5 mM MgCl2) at 200 rpm and 30 C. The next day, the EDC_LA\M 2245789\1 42 Date Recu/Date Received 2020-04-20 grown cells were centrifuged at 7,000 rpm for 10 minutes and centrifuged once more in the same manner as above. The collected supernatant was precipitated on ice in the presence of 1/5 (v/v) 20% PEG/2.5m NaCl of the supernatant for 1 hour.
After precipitation, centrifugation was performed at 7,000 rpm for 1 hour. The supernatant was discarded, and the precipitate was released with 3 ml of PBS, filtered through a 0.45 pm filter, stored at 4 C, and used in the next panning process. This process was repeated 3 to 4 times, and the antibody binding to the antigen was identified through ELISA.

[149]

[150] Example 2. Monoclonal ScFv- phage ELISA

[151] After the panning process was completed, the final round cell stock was diluted to form 200 to 500 colonies, spread on a CM agar plate, and then incubated overnight at 37 C. The next day, when the colonies grew, 200 pl of 2xYT
medium (CM 34 pg/ml + 1% glucose) was plated onto a 96-well deep plate, the colony was inserted into each well one by one and the plate was then incubated overnight at 37 C and 3,000 rpm. The next day, 200 pl of 2xYT medium (CM 34 pg/ml + 1% glucose) was plated onto a new 96-well dip plate, and 20 ml of cells grown the day before were injected into each well and grown at 37 C and 3,000 rpm for 1 hour and 10 minutes.
The remaining cells were stored at -70 C in 100 pl of 50%
glycerol. When the cells grew, 1 pl of a helper phage was EDC_LA\M 2245789\1 43 Date Recu/Date Received 2020-04-20 mixed with 19 pl of 2xYT medium, and 20 pl of the resulting mixture was injected into each well and incubated for 30 minutes at 37 C. After incubation, centrifugation was performed at 3,000 rpm for 10 minutes. The supernatant was discarded and 200 pl of 2xYT medium (CM 34 pg/m2 + Kan. 70 pg/m2 + 1 mM IPTG + 5mM MgCl2) was added to the residue, followed by incubation in MegaGrow at 30 C and 3,000 rpm overnight.

[152] 1 pg/ml of Ag (hVISTA-Fc, mVISTA-Fc) was plated at 100 pl/well onto a 96-well immunoplate and then incubated overnight at 4 C. The next day, the cells grown the day before were centrifuged at 3,000 rpm for 10 minutes and stored at 4 C. The plated Ag was washed 3 times with 0.05%
PBST, and then 200 pl of 2% BSA blocking buffer was added thereto, followed by incubation at 25 C for 2 hours. After blocking was completed, the result was washed 3 times with 0.05% PBST. 50 pl of 4% BSA was mixed with 50 pl of the phage stored at a down-regulated temperature of 4 C, and reaction therebetween was allowed to proceed while shaking at room temperature for 1 hour. After phage binding was completed, the reaction product was washed three times with 0.05% PBST, 100 pl of HRP-conjugated mouse anti-M13 antibody (1:3000, #GE 27-9421-01) was added thereto, and reaction was allowed to proceed at 25 C for 1 hour. After the reaction was completed, the resulting product was washed 3 times with 0.05%
EDC_LA\M 2245789\1 44 Date Recu/Date Received 2020-04-20 PBST, 100 pl of TMB (#BD TMB substrate reagent set 555214) was added thereto, color was developed for 3 to 5 minutes, 50 pl of a stop solution was added thereto, and then an assay was performed using an ELISA reader.

[153]

[154] [Table 13 Result of measurement by ELISA to select monoclonal scPv phage binding to VISTA antigen after panning process Ag : VISTA-FC OD, 1104 Clone- Hanaaw Mouse., 2117 2.0890 0.084a 2C12a 2.114 2.02110 11380 2.1350 0.30Z.
44, ID10a 2.0580 go, 1C94, 1.871a 0.0790 94, lAi 24, 1.8759 OAS&
3C'5, 7.2010 0.106

[155]

[156] Selected antibodies are shown in Table 2 below.

[157]

[158] [Table 23 EDC_LA\M 2245789\1 45 Date Recu/Date Received 2020-04-20 Antibody Amino Acid SequencsSE-Q ID NO
Heiwy-challo CDRI GGS&SNVA

Ileavy4bals CDILI AKGAIEPWPINFDS 3 IRS _____________________ Light-eke* CDRI NSDVGAVNY 4 Liskvelhais CDR2 DVI4 3 Lighicluolo CDR3 AAVVDDSINGIN
Ilkory-dola CORI GYIFIVIN 7 Reavyglida CDRI INPYDGRT
flon,y-dialsCURl AKQNIGIWDYDAFDI
: 2C12 Light-chain(DR I SSNIGAGI'D 10 Uglivebstr CDR2 GM II
LIght=ebela CDR3 VAWDDSLKAW 12 EDC_LAVV\ 2245789\1 4 6 Date Recu/Date Received 2020-04-20 Heavy-chain CDR1 GFSFHDYT 13 Heavy-cbaln CDR2 ISWDGTIT 14 Heavy-chain CDR3 AKKDRYDYYSGAFDI 15 1.412 Light-chain CDR1 SSDVGGYDY 16 Light-chain CDR2 DV'S 17 Light-chain CDR3 SSFAGSNTIRV 18 Heavy-chaln CDR1 GYTFSSYW 19 Heavy-chain CDR2 INPGNGHT 20 Heavy-chain CDR3 AKDIAYYDFWSGDAFDL 21 Light-chain CDR1 liLGINKY 22 Light chain CDR2 QDS 23 Light-chain CDR3 QTWDIFtSTGV 24 EDC_LAW 2245789 \ 1 47 Date Recu/Date Received 2020-04-20 [Table 3]
____________________ - ¨
Antibody Amino Acid Sequence SEQ ID 140 QMQINQSGAEVICICPCStiVKVSCKASCGSSSNY
AISIVVRQAPGQGLE'ANIGG I PIFG I I
VII SYAQICIWRVIITADGSNIS I AIME] ,I.,RSEDIAVYY 25 CAKG1,11:Pu PFVFDWGQG I [ATVS'S
188 __ QI.%=1 IvI,RSVSGSP4,,Q6. \ IIS( I NSDVGAYNY
VL I.sWyQQL (.14. \ PliVTITDVN
KR I"'( .1 I, DRFsGSRSGK I IISGLQAFDEADYYC 26 \ I) DSI,NGINIc .4. ( .T1.1:111 G
çJv VIA L=q.,1/ Kkl'4.1 E.\ s( KAMA II 11)1.1"
NUM RQAP(.QC;LEWAIGVItiPI-DGR I
VII SFAQI:FQGRI. I I itt 9 2C12 AKQMGIW DIDAFDIWGQG'IN TV 1% s s QFVLIQPSSVSl:APGQRVIIS( ,1(,SssN1(;A(;11'1) 'Ptvim QQLPta APICI'LlYGNS
2$
DRPSGVI'DRI s.µsKsATSASI.AITGIVAEDEADYlir N'tµ`µ1)1)st,ICAYVI't ,t t; I KV I VI.G
QMQLVI sGGGLVEPGRSLItL.S1 Ii SGFSF/IDYI
MIAVVRQVPGliGLEV1VSLIMVUGTIT
VII FVADPVRGRFIISRDNSICNSLYLQAINSLRAEIDTAVYY
C.
1Al2 ______ AKEDRYDYVSGAFDIWGQGTVVIIVSS
QSAILTQPRSVSGSPGQSVTISCTGISSDVGGYDY
VL
VSVVYQQHPGICAPICLILNDVN
KRPSGVPDRESGSICSGNTASLTISGLQPDDEAD'VYC
SsitAt ;AN 111.10. FGGG1KLYVLG
QVQLVQAIKIP SV SCKASCVITSSVW

N11 7i1t74.1EKFKSRVTITVDICs ksTAYNIELSSLItSEDTAVVVC 31 AIi.DIAVyppvs(41,44111..VitG429. nIVIViSS
SVELIQPISVSVSPGQIASITCSCUKLGNICV
ASAVYQQKPCQSPVLVIVIPN

KRPSGIPERFSGSNSGNIATLTISGTQATDEADYYC
QTWORSICVFIGICINVTVIS;

[159] Example 3. Binding of anti-VISTA ScFV phage to VISTA
expressing CHO -K1 cells

[160] CHO -K1 cells expressing human VISTA and mouse VISTA
were produced in order to determine whether or not the screened antibody binds to cells expressing VISTA. 50 pl of the produced cells were plated onto a 96-well dip plate at a EDC_LAW\ 2245789\1 48 Date Recu/Date Received 2020-04-20 density of 4x106 cells/ml in FACS buffer (2% FBS, 0.05% sodium azide in PBS), 50 pl of phage supernatant undergoing phage incubation was added thereto, and was incubated at room temperature for 1 hour. After incubation, 400 pl of FACS
buffer was added to each well, followed by centrifugation at 2,000 rpm for 5 minutes. The supernatant was discarded and 100 pl of anti-M13 antibody (1:1000) was added to the residue, followed by reaction at 4 C for 1 hour. After the reaction was completed, 400 pl of FACS buffer was added to each well, followed by centrifugation at 2,000 rpm for 5 minutes. The supernatant was discarded, and 100 pl of anti-mouse IgG-PE
antibody (1:500) was added thereto, followed by reaction at 4 C for 1 hour. After the reaction was completed, 400 pl of FACS buffer was added to each well, followed by centrifugation at 2,000 rpm for 5 minutes. The supernatant was discarded, and 200 pl of FACS buffer was added thereto, followed by assay using FACS (Beckton Dickinson, FACSCalibur).
The results are shown in FIG. 1.

[161] As can be seen from FIG. 1, all of the selected antibodies bound to the CHO-K1 cells produced to overexpress human VISTA. The 6A6 used as a negative control group did not bind thereto and the VISTA antibody (R&D systems #FAB71261A) used as a positive control group bound to cells well, as expected.

[162]
EDC_LA\M 2245789\1 4 9 Date Recu/Date Received 2020-04-20

[163] Example 4. Anti-VISTA IgG expression

[164] Conversion of the screened scFv phage to the IgG form was performed using molecular biology techniques. Phagemid was extracted from the screened E. Co/i clone and double-cut with restriction enzyme Sfi I (R0123, New England Biolabs, US) to obtain a heavy-chain variable-region DNA fragment, and a pIgGHD-6A6Hvy vector including a heavy-chain constant region was treated with Sfi I, and the variable-region DNA
fragment was then inserted. In the same manner as above, the phagemid was double-cut with the restriction enzyme BstX I
(R0113, New England Biolabs) to obtain a light-chain variable region DNA fragment, a pIgGLD-6A6Lgt vector including a light-chain constant region was double-cut with BstX I, and the variable-region DNA fragment was inserted to complete DNA cloning into an IgG form.

[165] The transient expression of IgG was performed using an Expi293F expression system kit (Thermo Fisher Scientific, US). Expi293 cells included in the kit were subjected to suspension culture on a 125 rpm orbital shaker at 37 C and 5% CO2 using a dedicated medium. Every 3 days, the cells were passage-cultured to 3 x 105 cells/ml, and when an expression vector was introduced, the number of cells was adjusted to 3 x 106 cells/ml before use. The gene introduction was performed using, as a dedicated reagent, ExpiFectamine, and EDC_LA\M 2245789\1 50 Date Recu/Date Received 2020-04-20 a Lipid-DNA complex containing 1 pg of an expression vector DNA and 2.7 pl of ExpiFectamine per 1 ml of a cell suspension was prepared and added to the cell suspension. 16 to 18 hours after the introduction, the half M of an enhancer was added to induce expression. Then, the cells were cultured under the same conditions as above for 3 to 4 days and centrifuged to obtain a supernatant containing IgG.

[166]

[167] Example 5. Purification of anti-PD-Li antibody

[168] The obtained supernatant was injected into a protein A column (GE Healthcare) and IgG was purified through affinity chromatography. After the column was equilibrated with 20 mM Tris-HC1, 50 mM NaCl, and 5 mM EDTA (pH 7.0), the supernatant was injected, washed with 50 mM Tris-HC1, 500 mM
NaCl, 5 mM EDTA, and 0.2% polysorbate 20 (pH 7.0) solution, eluted with 50 mM NaCl, 0.1 M glycine-HC1 (pH 3.5) and neutralized with 1 M Tris. The eluted protein was dialyzed using MWCO 10,000 spectra/por dialysis membrane (Spectrum Labs, US) to replace the solvent with PBS. Then, the protein was concentrated to the required concentration using Vivaspin (Sartorius, DE), dispensed and stored at -80 C.

[169] After purification, each antibody was treated with non-reducing and reducing LDS sample buffers (Thermo Fisher Scientific) and electrophoresed using a NuPAGE System (Thermo EDC_LA\M 2245789\1 51 Date Recu/Date Received 2020-04-20 Fisher Scientific). As a result, IgG having a total molecular weight of about 150 kDa including a 50 kDa heavy chain and a 25 kDa light chain was obtained.

[170]

[171] Example 6. Binding ability of anti-VISTA antibody to antigen

[172] The binding ability of the screened antibody to the antigen was measured using an Octet (ForteBio). For this purpose, the antibody was immobilized on a biosensor, various concentrations of antigen were added to a 96-well plate, and then the binding ability was measured.

[173]

[174] [Table 43 Results of determination of binding ability of various concentrations of anti-VISTA antibody to VISTA
antigen in order to measure binding ability therebetween kon(11Ms) kdis(lis) KO (M) #287 2A5E+04 142E-04 5.80E09 #2C12(H) 8.34E+04 8A8E-05 102E-09 #2C12(M) 5.57E+04 7.80E44 140E48 #188 1.11E+05 1.03E-03 9.29E-09 #1D10 4.17E4-04 1.81E-04 4.34E-09 #1C9 2.33E+05 7.30E04 113E-09 #1Al2 5.02E+04 234E05 5.46E-1O
#3C5 2_64E+05 3.67E46 139E-08 EDC_LA\M 2245789\1 52 Date Recu/Date Received 2020-04-20

[175] Example 7. Cross-reactivity of anti-VISTA antibody to human and mouse VISTA

[176] ELISA assay: 1 pg/m1 of Ag (human VISTA-Fc or mouse VISTA-Fc) was plated at 100 pl/well onto a 96-well plate and incubated at 4 C overnight. The next day, the plated Ag was washed 3 times with 0.05% PBST and 200 pl of 2% BSA blocking buffer was added thereto, followed by incubation at 25 C for 2 hours. After blocking was completed, the resulting product was washed 3 times with 0.05% PBST. 1 pg/m1 of each antibody was injected at 100 pl/well and incubated at 25 C for 1 hour.
After incubation was completed, the resulting product was washed 3 times with 0.05% PBST and 100 pl of goat anti-human IgG (kappa)-conjugated peroxidase (1:2000, Bethyl Lab # A80-115P) was added thereto, followed by incubation at 25 C for 30 minutes. After incubation, the resulting product was washed 3 times with 0.05% PBST, 100 pl of TMB (BD TMB
substrate reagent set # 555214) was added, color was developed for 3 to 5 minutes, 50 pl of a stop solution was added thereto, and then an assay was performed using an ELISA
reader. The results are shown in FIG. 2.

[177] As shown in FIG. 2, all of the screened antibodies bound to human VISTA, in particular, the 2C12 antibody bound well to mouse VISTA as well as human VISTA.

[178] FACS assay: to identify overexpression of human and EDC_LA\M 2245789\1 53 Date Recu/Date Received 2020-04-20 mouse VISTA in CHO-K1 cells, a cell solution (5X105 cells/100 pl/FACS tube) at a concentration of 5x106/m1 in FACS buffer (2% FBS, 0.05% sodium azide in PBS) was prepared, 2 pg/ml of the screened antibody was prepared, and 100 pl of the cell solution and 100 pl of the antibody were placed in a FACS
tube and then incubated at 4 C for 30 minutes. After incubation was completed, 2 ml of FACS buffer was added to each tube, followed by centrifugation at 1,500 rpm for 5 minutes. The supernatant was discarded, 100 pl of a goat anti-human IgG-Fc-conjugated PE (A80-248PE, 1:500) was added to each tube, and a reaction was allowed to proceed at 4 C
for 20 minutes. In the case of a positive control group, 5 pl of APC-conjugated anti-hVISTA (R&D #71261A) was added to hVISTA over-expressing cells (in the same amount as sample) and in the case of mice, 1 pl of APC-conjugated anti-mVISTA
(BioLegend #143710) was added to mVISTA over-expressing cells (in the same amount as the sample), followed by reaction at 4 C for 20 minutes. When the reaction was completed, 2 ml of FACS buffer was added to each well, followed by centrifugation at 1,500 rpm for 5 minutes. The supernatant was discarded, and 200 ii of FACS buffer was added thereto, followed by an assay using BD FACSCalibur. The results are shown in FIG. 3.

[179] As shown in FIG. 3, when treating CHO-Kl cells prepared to overexpress VISTA with the screened anti-VISTA, EDC_LA\M 2245789\1 54 Date Recu/Date Received 2020-04-20 like in ELISA, all screened antibodies bound to CHO-K1 expressing human VISTA on the surface thereof and the 2C12 antibody also bound to CHO-K1 expressing mouse VISTA on the surface thereof.

[180] Example 8. Allo-MLR assay of anti-VISTA antibody

[181] 50 ml of human peripheral blood was collected from a donor and diluted 3-fold with PBS containing 2 mM EDTA. 15 ml of Ficoll -Paque (GE #17-1440-03) was injected into a fresh 50 ml tube, and 30 ml of diluted blood was slowly loaded, followed by centrifugation at 2,000 rpm for 40 minutes. The PBMC layer was recovered, transferred to a 50 ml tube and washed with washing buffer (RPMI1640 medium containing 2% FBS and 10 mM HEPES) by centrifugation at 1,600 rpm for 10 minutes. After centrifugation, the supernatant was discarded and washed repeatedly. The cell pellets left after centrifugation were released into single cells with 50 ml of washing buffer, and then a dead cell mass was removed using a cell strainer (40 um). The cells (donor A or B) to be used as responders of Allo-MLR were diluted to a concentration of 4 x 106 cells/ml with MLR medium (RPMI1640 medium containing 10% FBS, 10 mM HEPES, 1 mM pyruvate, NEAA
(100X), 2 mM L-glutamine, 1% antibiotics, 10-5M p-ME). Cells (donor B or A) to be used as stimulators were treated with mitomycin C (MMC) to inhibit cell proliferation. The method will be described Date Recue/Date Received 2021-06-29 in detail as follows. Cells to be used as stimulators were diluted in MLR medium at a concentration of 4 x 106 cells/ml in a 50 ml tube, and MMC was added to reach a total volume of 25 pg/ml, followed by reaction for 30 minutes at 37 C.
After the reaction was completed, a 50 ml tube was completely filled with a washing buffer and centrifugation was performed at 1,200 rpm for 10 minutes, followed by washing. This process was performed 4 times to completely remove MMC.
Finally, the residue was diluted in MLR medium to a concentration of 4 x 106 cells/ml. The prepared cells were aliquoted at 2 x 105 cells/well (50 pl) into a 96-well cell culture plate. 50 pl of the antibody to be analyzed was added to realize final concentrations of 30 pg/ml and 10 pg/ml.
The antibody was tested using 3 wells for each concentration.
After incubation for 5 days in a CO2 incubator at 37 C, 50 pl of the cell culture supernatant was stored separately for measuring IFN-y production, and the remaining cells were used for analysis of cell proliferation. The cell proliferation was analyzed by measuring the amount of ATP produced in cells left after allo-MLR. The method is described in detail as follows. 100 pl (equivalent to cell culture solution) of CellTiter-Glo Luminescent cell viability assay (Promega) solution was added to the cultured cells and reacted for 10 minutes at room temperature to induce lysis of the cells.
Cell viability was measured using a luminometer. The results EDC_LA\M 2245789\1 56 Date Recu/Date Received 2020-04-20 are shown in FIG. 4.
[183] As shown in FIG. 4, the competitors (positive control group) and 1B8 clones induced expression of more interferon-gamma than the group treated with non-specific antibody (whole IgG), and all other clones induced lower interferon-gamma expression than whole IgG. In other words, these clones are clones that suppress the expression of interferon-gamma. Among them, 2C12, 1Al2 and 3C5 exhibited excellent interferon-gamma expression suppression ability.
[184]
[185] Example 9. Anti-tumor activity of anti-VISTA
antibody [186] The anticancer efficacy of caudal vein administration of human PBMCs and JNJ61610588, pembrolizumab and anti-VISTA mAb 1B8 therapeutic agents was determined using NSG mice including pads transplanted with a human-derived breast cancer cell line, MDA-MB-231 mammary fat, to evaluate the anti-tumor activity of the anti-VISTA
antibody. The experiment was conducted jointly with Laboratory Animal Resource Center, the Korea Research Institute of Bioscience and Biotechnology.
[187] The human breast cancer cell line, MDA-MB-231, was thawed and cultured in a CO2 incubator (Forma, USA) at a temperature of 37 C and a CO2 concentration of 5%. On the EDC_LA\M 2245789\1 57 Date Recu/Date Received 2020-04-20 final day of culture, all cancer cells were collected and counted, and the cell concentration was adjusted to 3 x 107 cells/ml using serum-free media. The cell culture solution thus adjusted was injected at 0.1 ml (3 x 106 cells/mouse) into each of left and right regions of the mouse breast fat pad.
[188] After transplantation with the cancer cells, when the average tumor size for each group reached 46.9 mm3, PBMC
(4x107 cells/ml) of A type (No. 1 mouse in each group), B
type (No. 2 mouse in each group), C type (No. 3 mouse in each group), D type (No. 4 mouse in each group), E type (No.
mouse in each group) and F type (No. 6 mouse in each group) was injected at 0.16 ml (0.64x107 cells/mouse) through caudal vein administration.
[189] For PBMC separation, 50 ml of human peripheral blood was collected from a donor and diluted 3-fold with PBS
containing 2 mM EDTA. 15 ml of Ficoll-Paque (GE #17-1440-03) was injected into a fresh 50 ml tube, and 30 ml of diluted blood was slowly loaded therein, followed by centrifugation at 2,000 rpm for 40 minutes. The PBMC layer was recovered and transferred to a 50 ml tube, and the cells were washed with a washing buffer (RPMI1640 medium containing 2% FBS and mM HEPES) by centrifugation at 1,600 rpm for 10 minutes.
After centrifugation, the supernatant was discarded and washed four times. The cell pellets left after centrifugation EDC_LA\M 2245789\1 58 Date Recu/Date Received 2020-04-20 were released into single cells with 50 ml of washing buffer, and were then diluted to a concentration of 2 x 107 cells/mL
in PBS medium.
[190] For the organization of the mouse group and administration of drug into the mouse group, after PBMC
injection, drug administration was started. The drug was administered through the caudal vein at 5 mg/kg into each group of Group 1: PBMC + human whole IgG (control); Group 2:
PBMC + competitor (JNJ 61610588); Group 3: PBMC +
pembrolizumab; Group 4: PBMC + anti VISTA mAb(1B8); and Group 5: PBMC + pembrolizumab + anti VISTA mAb(1B8).
[191] After PBMC injection, the drug was first administered at 0.2 ml per mouse twice a week for a total of 7 times.
[192] All animals were observed for general clinical symptoms and changes in body weight and tumor size at the beginning of administration and immediately before each administration during the experimental period.
[193] The change in tumor size was expressed using the formula length x width x height/2 after measuring left and right tumors in three directions using a Vernier caliper for each of 12 subjects from the time at which the average tumor size of each cancer cell transplant group reached 46.9 mm3 until the 25th day thereafter.
[194] On the 25th day from the beginning of drug EDC_LA\M 2245789\1 59 Date Recu/Date Received 2020-04-20 administration, 3 healthy mice per group were selected, blood was collected from the orbital veins thereof (EDTA tube), the mice were sacrificed using CO2 gas, the spleen was extracted and immobilized in PBS, and the tumors were divided into right and left sections. The tumors were weighed using a chemical balance and then imaged and immobilized in liquid nitrogen and formalin.
[195] As a result, no administration groups exhibited specific general symptoms during the test period, but the solvent control group (PBMC + PBS) No. 2 mouse and the PBMC
+ Anti-VISTA mAb 1B8 administration group No. 5 mouse showed weight loss from the 17th day and the 21t day, respectively, to the last day, compared to the initial weight thereof (Table 5 and FIG. 5).
[196] [Table 5] Weight change of mouse on each day (%) Group Dose Days after treatment (n=6) (ugiity) Day 0 3 5 7 10 12 14 17 19 PBMC+PBS 100.0 1024 104.7 1073 110.4 112.2 115.5 117.3 1113.9 120.0 120.6 122.0 (Solvent control groj 0.0 t0.3 0.5 *1.3 +2.7 +15. 4.2 +8.0 6.9 8.6 +11.5 +13.1 PBMC 100.0 102.0 104.1 106.6 109.6 112.0 113.6 116.6 116.4 121.5 126.6 128.8 -WM.161610588 *0.0 1.3 *1.6 2.4 4,0 *4.0 *3.9 *3.4 *3,7 13.3 14,1 *3.7 PBMC
100.0 101.5 103.6 106.1 108.9 1114 112.8 114.4 913.1 114.3 114.5 116.2 +Pernbrollizuma 5 0.0 *1.9 *2.5 *2.1 3.0 *2.8 *4.1 *3.6 *4.6 5.3 *6.9 . 5.3 PBMC
100.0 101.7 103.7 107.2 109.7 1.132 114.5 118.2 120.6 122.1 123.6 125.4 +Anti-VISTA 5 0.0 1.1 *1.4 2.2 *2.5 *2.4 *3.6 5.0 *5.2 *6.5 *11.9 13.6 mAb 1B8 PBMC
100.0 101.2 103.3 105.8 108.8 110.0 112.3 114.2 113.6 115.5 1174 119.9 5+5 +PbrIzm+Anti-0.0 +1.5 11.7 +1.9 2.1 2.0 1.9 3.2 *3.1 3,5 t4.3 *3.5 VI mAb B8 EDC_LAVV\ 2245789\1 60 Date Recu/Date Received 2020-04-20 [197] On the 25th day after the beginning of drug administration, the MDA -MB -231 tumor was excised and weighed.
As a result, PBMC + JNJ61610588 (5 mg/kg), PBMC +
pembrolizumab (5 mg/kg), PBMC + Anti-VISTA mAb 1B8 (5 mg/kg) and PBMC + Pembrolizumab + Anti-VISTA mAb (5+5 mg/kg) administration groups exhibited statistically significant reductions in tumor weight of 18.9% (p<0.05), 32.9% (p<0.001), 37.8% (p<0.001) and 60.6%(p<0.001), respectively (Table 6, FIG. 6), compared to the solvent control group (PBMC + human whole IgG).
[198] [Table 6] Change in tumor size and tumor weight on last day Tumor Group Dose Tumor volume( Vt-vot weight õ (mg) (n=6, Left+right), o 3 6 7 10 12 14 17 19 21 24 26 26 PBMC+PB 67.2 128. 225. 317. 440. 572. 688.
837, 964.
36.8 0.0 16.5 6 5 3 +15 4 4 4 8 0 1110.5 0 +10 (Solvent contre1.0 12.6 2 22 135 151 171. +96 +10 +14 115 ..

.1 .
.9 .4 .1 0 5 7.5 6.5 8.2 grmip) 26.2 49.8 94.6 163. 239., 330. 429. 526. 668. 782.
0.0 .1 2 7 2 8 1 0 5 900.8 19. 18 127 PBMC *57 1:79 188,1,10 111 115 21 247.9 .1( 5.3 +..IW61610 5 1.0 0 .6 .2 .3 .6 6 4.7 5.9 5.0 8.9 = = or.
588 == == == = =
26.5 28.9 25.9 26.4 27.6 2.5 25.0 24.9 23.4 20.3 18.8 18.V%
%s % % % % % % % % % %
35.2 106. 181. 249, 311. 332. 473. 585. 456.
13.4 30.3 7 5 3 9 8 2 5 2 744.9 0.0 .111 :LIB 137 150 156.173. 174. /84. 14 1163.0 PBMC t0.0 .0 ** 1 .3 4 2 2 2 1.2 +Pembroli zumab 16.0 17.0 17.8 17.8 193 21.4 29.2 31.4 31.3 32.1 31.9 32.1%
% % % % % %% O,, %% %
27.6 49.9 164, 226. 276. 353. 424, 527, 589.
93.9, 13.6 3 4 3 7 5 5 4 691.2 PBMC 0.0 16. 111 121 36 53 166.187. 96. 11 117 1208.1 +Anti- 10.0 13.2 6 .6 .8 5 .3 .6 3 0 6 5.7 3.8 VISTA
41. .1. 4.4r Ir.
mAb 1B8 16.0 25.0 25.7 28.6 27.2 2%0 36.8 301.2 30.3 37.0 37.8 37.6%
% % % % % % % % % %
22,9 42.0 78.8 128. 153. 184. 232. 276. 340. 378.
10.6 1 1 9 2 7 6 5 437.8 0.0 +6. 114 PBMC 0.0 t 3.0 3 +18 -131 142 149. 158.169. 180.
/95. i 98.1 .3 .1 +Pbr re. izm+A 5+5 J .5 0 8 6 6 0 nii-VImAb 188 36.11 37.7 37.5 38.7 43.2 $1.13 53.0 594 nye 59.3 60.7 60.6%
% % % % % % % % %
EDC_LAW\ 2245789\1 61 Date Recu/Date Received 2020-04-20 Significant figures(t-TEST) : * p<0.05, ** p<0.01, *** p<0.001 (vs PBMC+PBS) tAt=Vt-Vo, Vt(Measurement of the tumor volume), Vo(Initial tumor volume) Inhibition Rate (vs PBMC+PBS) [199]
[200] Example 10. Production and screening of variants to enhance affinity [201] Antibody optimization was performed to enhance the affinity of the anti-VISTA antibody clone 1B8. A primer having random mutations introduced into the light-chain CDR3 and heavy-chain CDR3 of 1B8 was produced using a soft-randomization method that preserves 80% of the original DNA
sequence of 1B8 and randomizes the same. A DNA fragment encoding a light-chain variable region of 1B8 and a heavy-chain variable region of ME4 into which a mutation was introduced was obtained through PCR using the primer. Each of the light-chain variable region and the heavy-chain variable region of the 1B8 scFv phage phagemid was replaced with the DNA fragment to produce a 1B8 light-chain CDR3 variant scFv phage library and a heavy-chain CDR3 variant scFv phage DNA library, respectively.
[202] The variant scFv phage DNA library was purified using phenol-chloroform and then transformed into an E. coli strain, EDC_LA\M 2245789\1 62 Date Recu/Date Received 2020-04-20 XL-1 Blue, using electroporation. Diversity acquisition was identified through transformation efficiency analysis and DNA sequence analysis, phage expression was induced by culturing at a 500 ml scale, and a 1B8 light-chain CDR3 variant scFv phage library was produced using a PEG-precipitation method.
[203] Biopanning was performed using each variant scFv phage library in the same manner as in Example 1. Then, in the screening process, the dissociation rate constant (kdis) of scFv was measured as a quantitative index for evaluating the binding retention ability. The result showed that 7 screened optimized clones retained the heavy-chain variable region of 1B8. The result of measurement of the dissociation rate constants (Table 7) and the amino acid sequence (Tables 8 and 9) of the 7 screened optimized clones are shown.
[204] The result of measurement of dissociation rate constants of the seven optimized anti-VISTA antibodies showed that the PMC-309.3.3 sequence was obtained by substituting N
(Aps) of the CDR3 portion of the best anti-VISTA.H2 with Y
(Tyr). The result of comparison and analysis of the sequence of PMC-309.3.3 with a human germline base sequence using IMGT
software (http://imgt.cines.fr/IMGTvquest/share/textes/) showed that the heavy-chain variable region was derived from IGHV1-69*06. The result of comparison with the amino acid EDC_LA\M 2245789\1 63 Date Recu/Date Received 2020-04-20 sequence of IGHV1-69*06 of the germline group showed that 4 amino acid residues of the framework region (FR) were substituted. In order to increase the folding efficiency while maintaining antigen-binding ability, PMC 309.3.4 with increased efficiency through substitution of the FR portion was produced. The specific sequence of PMC 309.3.4 is as shown in Table 10.
[205] [Table 7]
Phagedone Kdis(1/s) Anti VISTA.3.0 (1B8) 7.32E-04 Anti VISTA B5 6.16E-04 Anti VISTA G1 5.74E-04 Anti VISTA H2 (1) 2.85E-04 Anti VISTA V3 1 4.87E-04 Anti VISTA V3 2 1.08E-03 Anti VISTA V3 3 1.27E-03 Anti VISTA V3 4 2.22E-03 [206] [Table 83 Amino acid sequence SEQ ID No.:
Anti VISTA B5 VL CDR3 AAWDDDLNGLV 33 Anti VISTA G1 VL CDR3 AAWDDSLIGHV 34 Anti VISTA H2 VL CDR3 AAWDDNLNGLV 35 Anti VISTA_V3 1 VL CDR3 AAWDDSLYGLV 36 Anti VISTA_V3 2 VL CDR3 AAWDDSLNDLV 37 Anti VISTA_V3 3 VL CDR3 AAWDDSLNVLV 38 Anti VISTA_V3 4 VL CDR3 AAWDDNSDFHV 39 EDC_LA\M 2245789\1 64 Date Recu/Date Received 2020-04-20 PMC309.3.3 I VL CDR3 I AAWDDNLYGLV I 40 [Table 9]
Name Hrl(111) Lgt(1,2) QINLTQPRSVSGSPGQSVTISCTGINSDVGAYNYLSWYQQLPGR.APK
VIIYDVNKP.PSGVPDRFSGSRSGKTASLTISOLQAEDEADYYCAAWDD
Anti 11S'EA BS DLINGLVFGGGTKLTVLG
SEQ ID NO.: 41 QLVLTQPRSVSGSPOQSVTISCIGINSDVGATNYLSWYQQLPGRAPK
VIIYDVNZKRPSGVPDRFSGSRSGKTASLTISGLQAEDEADYYCAAWDD
Anti VISTA_G1 SLIGHVEGGOTKLTVLG
SEQ ID NO.: 42 QINLTQPRSVSGSPOQSVTLSCTGTNSDVGAYNYLSWIQQLPGRAPK
Anti VISTA_H2 VIIYDVNKRPSGVPDRFSGSRSGKTASLTISGLQAEDF_ADYYCAAWDD
NIAGINFGGGTKLTVLO
SEQ NO.: 43 QMQLVQSGAEVKKPGSSV QLVLTQPRSVSGSPGQSVTISCTGTNSDVGAYN. YLSWYQQLPGR_APK
And V1STA_V3_1 KVSCKASGGSSSNYAISWV
NIIYDVNXRPSGVPDRFSGSRSGKTASLTISGLQAEDE.AD'rfCAAWDD
RQAPGQGLEWMGGIIPIF SLVGLVFGGGTKLTVLG
GTTSYAQKFQGRVTIT.ADG SEQ ID NO.: 44 SMSTAYMELSSLRSEDTAV QINLTQPRSVSGSPGQSVTISCTGTNSDVG.AYNYLSWYQQLPGR.APK
YITAKGAIEPANTFVEDN VIIYDVNKRPSGVPDRFSGSRSGKTASL TISGLQAEDEADYYCAAWDD
Anti 11ST.A_V3_2 WGQGTIVTVSS SLNDINFGGGTKLTVLG
SEQ ID NO.: 25 SEQ ID NO.: 45 QINLYQPRSVSGSPOQSVTISCTGTNSDVGAYNIISWIQQLPGRARK
1.11YDVNKRPSGVPDRISGSRSGKTASLTISGLQAEDEADINCAAWDD
And VISTA 173_3 SLNVIATGOGTKLTVLG
SEQ ID NO.: 46 QINLTQPRSVSGSPGQSVTISCTOTNSDVGAYNTLSWYQQLPGRAPK
A 11STA V3_4 VIIYDVNKRPSGVPDFtFSGSRSGKTASLTISGLQAEDEADYYCAAWDD
nti _ NS DFITVFGOOTKLTVLG
SEQ ID NO.: 47 QLVLTQPRSVSGSPGQSVTISCTGTNSDVGAYNAISVVYQQLPGRAPK
VIIYDVNKRPSGVPDRFSGSRSGICT.ASLTISGLQAEDEADYWAAWDD
PN IC34)9.3.3 NLYC,L\TGGGTKLTVLG
SEQ ID NO.: 48 [Table 10]
name Hvy Lgt Hvv sea. 141t wt.
QLVLTQPRSVSGSPGQSVT1SC'TGTNSDVGAYNYL.SWYQ
QVQLVQSGAEVICKPGSSVICVSC
QLPGRAPKVIIYDVNILItPSGVPURFSGSRSGKTASMSG
PMC309.3.4-W G-H wt Lgt KASGGSSSNYA1SWVRQAPGQG
LQARDEADYYCAAWDUNLIGLVFGGGTILITVLG
LEWMGGIIPIFGTTSYAQKFQG SEQ ID No.: 48 RVTTIADKSTSTAYNIELSSIASK
QSALTQPRSVSGSPGQSVTISCIGTNSDVGAYNVLSWYQ

QLPORAPICVHYDVNkItPSGVPDRFSGSRSGIKTA.SLTISG
PMC309.3.4-SA G-H SA. GQGTIVTvSS
LQAEDRADINCAAWDDNLVGINFGOGTKIYVI, SEQ ID No.: 49 SEQ jp No.:, 50 [209] Example 11. Analysis of physicochemical properties EDC_LAW\ 2245789\1 65 Date Recu/Date Received 2020-04-20 of optimized anti-VISTA antibody [210] Each optimized scFv phage clone was converted to an IgG form. Specific DNA cloning, production, and purification methods for the form conversion were performed using the same methods as those described in Examples 4 and 5.
[211] Octet was used to evaluate the binding strength of the optimized anti-VISTA antibody while excluding clones with low productivity therefrom, and the experiment was performed in the same manner as the method presented in Example 6 (Table 11). As can be seen from Table 11, the binding force to human VISTA was significantly increased through the optimization.
[212]
[213] [Table 11]
TM
Lipid 1 Anayte (M) kon(1:11.9 kdisatt) Scatter Scatter Ratio-H1 Anti VISTA
AE-84 1.54E+05 2.09B-03 68.98 70.93 7 1..03 =
3.0(1BS) Anti VISTA
2.86E-09 5.257:404 1 50E-04 70.28 71.63 74.76 Anti NISTA
1 28E-CS 1.4905 1.91E-03 62.58 65.15 66.50 V3' VIS T A ______________ I PMC-309.3.3 16E-10 1.85E-05 9.56E-05 68.53 69.26 "2.10 PMC 309.3.-1 L6E :0 1.89-05 9.E.05 69.05 70.15 .PMC-3093,4SA I 6 06E- 10 2.25E-05 1.34E-04 62.05 66.46 67.34 [214]
[215] Example 12. Allo-MLR assay of optimized anti-VISTA
antibody EDC_LA\M 2245789\1 66 Date Recu/Date Received 2020-04-20 [216] In order to evaluate the function of the optimized antibody, a mixed lymphocyte reaction (MLR) was performed in the same manner as in Example 8 to measure the reactivity of immune cells contained in peripheral blood in vitro.
Thereby, it was confirmed that cell proliferation and cytokine (IFN-y) production were induced.
[217] The results are shown in FIGS. 7 and 8. As can be seen from FIGS. 7 and 8, the PMC-309.3.3 drug was very superior to the comparative group and that concentration-dependent cell proliferation and cytokine secretion increased.
[218]
[219] Example 13. Anti-tumor activity of optimized anti-VISTA antibodies [220] In order to evaluate the anti-tumor activity of the anti-VISTA antibody, the anticancer efficacy of caudal vein-administered drugs (PMC-309.3.4W, PMC-309.3.45A) having changed light-chain variable regions, including human PBMC
and JNJ61610588, PMC-309.3.3, and anti-VISTA 3.4, was evaluated in the same manner as in Example 7 using NSG mice including pads transplanted with a human breast cancer cell line, MDA-MB-231 mammary fat.
[221] As can be seen from Table 12, Table 13, and FIGS. 9 and 10, the result showed that PMC-309.3.3 showed the best EDC_LA\M 2245789\1 67 Date Recu/Date Received 2020-04-20 anticancer activity, which corresponds to the result in the mixed lymphatic reaction.
[222]
[223] [Table 123 Days atter treatment Group Dose (n=6) (mg4q) 0 1 3 6 B 10 13 15 17 20 22 Wventcontrol 100 0 100 5 101.8 104.2 107.0 107.8 109 8 110.7 111.9 112.7 115 7 117.2 118.5 118.9 grOup P8MC 5 0.0 11 0.9 /1.6 13.3 t3.6 t3.8 4.9 t5.9 *7.3 15.9 5.4 *5,1 *5.7 whole l'aG
PENG 6 160.0 1110.1 101.5 103.8 105 f 106 108 3 109.0 111 2 112.3 114.7 116.5 117.5 1180 41/876112 0.0 *1.1 1.6 11.8 12.3 t3.2 12.6 t3.4 *3.6 :4.3 *3.9 14.1 t5.0 1;5.3 MMIC 1000 100.6 101.6 103.8 106.9 107.3 108.9 109.6 112.1 114.1 116.6 118.8 120.8 121.3 +PMC309.3.3 *0.0 *1.2 *1.2 t1.5 *2.3 *3.2 *3.8 *5.1 t4.8 *4.6 33.9 14.1 *3.7 *3.2 PBMC 100.0 93 8 100.0 102.7 104.4 105.0 107.0 106.8 109.0 110.6 112.7 115.8 1182 118.3 PMC309.3.4W 0.0 11 /1.3 11.2 11.4 12.0 +.2.2 2.8 *1.8 1.9 1.5 1.2.8 +2.7 1.8 PEAK 100.0 99.5 100.7 102.7 104.3 104.0 105.9 105.8 108.1 109.3 111.8 114.2 116.5 117.4 -..pmC309 3 4SA 0.0 1 2 08 1 4 2 3 2 5 2.7 3.0 3.3 3 7 3.13 13.6 4.2 16.2 [224] [Table 13]
GrouP TUMOf volume(Vt-Vo)t Tumor wftWM9 04. Dose 14fOrl9b1) 059110) 0ey0 3 6 8 10 13 15 17 20 22 24 MAC
.whole IgG 0.0 20.6 56.7 94.8 138.9 256.1 383.5 477.8 633.4 787.8 998.0 1230.5 1428.5 15002 (solvent control 10.0 *4.5 9.7 3%1 t21.5 344.4 1592 a196 92.5 /103.8 148.2 3173.0 t222.3 1233.8 group) 0.0 148 40.6 71.6 1052 192.7 273.3 364.4 490.3 618.6 788.9 940.9 1167.0 1237.8 PBMC *0.0 *3.5 38.1 8.9 314.5 *27.3 *312 145.8 *59.4 188.1 t80.5 t95.8 1179.8 *191.6 28.1%, 28.4% 24.4% 24.2% 24.7% 24.8% 23.7% 22.6% 21.5% 21.0% 20.3% 18.2%
ma%

PI3MC tO 0 +36 11 +13 +21 3 +29 2 +48 4 Ã3.5+ +99 3 11113 +144 9 +117 5 +222 4 *2248 5 .
PMC309 3.3 42.0% 46.5% 45.1% 48.9% 47.5% 48.8% 49.8% 50.4% 50.7% 51.0% 51.1% 51.9%
50.8%
0.0 13.0 33.5 59.1 85.4 157.7 221 1 291.0 381.6 476 9 612.0 769.8 896 3 951.5 PBMC 0.0 *3.4 8.0 +16.8 +23.8 48.8 631 /92.3 98.8 133.0 157.9 177 I +.242.4 *244.7 iPMC.309.3.4W
38.8% 40.9% 37.6% 38.5% 38.4% 39.2% 39.1% 39.7% 39.5% 38.5% 37.4% 37.2%
37.0%
0.0 13.4 33.9 629 92.5 169.2 233.5 319.0 415.8 524.0 663.4 812.1 944.9 1034.3 PBMC 0.0 +2.6 5.4 12.7 +20.2 40.7 42.3 366.4 *87.9 121.9 151.3 1776 198.3 199.9 PMC309.3 4SA
34.7% 40.3% 33.7% 33.4% 33.9% 35.8% 332% 34.4% 335% 33.4% 34.0% 33.8% 33.5%
EDC_LAVV\ 2245789 \ 1 68 Date Recu/Date Received 2020-04-20 Significant figures(t-TEST) : * p<0.05, ** p<0.01, *** p<0.001 (vs PBMC+whole IgG) tAt=Vt-Vo, Vt(Measurement of the tumor volume), Vo(Initial tumor volume) 1 Inhibition Rate (vs PBMC+ whole IgG) [225] Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this description is provided to set forth preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalents thereto.
[226] [Sequence Listing Free Text]
[227] An electronic file is attached.
EDC_LA\M 2245789\1 69 Date Recu/Date Received 2020-04-20

Claims (9)

Claims

1 . An antibody or an antigen-binding fragment thereof binding to V-domain Ig suppressor of T-cell activation (VISTA) comprising:
a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 6, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 33, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 34, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 35, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a Date Recue/Date Received 2021-06-29 heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 36, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 37, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 38, a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 39, or a heavy-chain variable region comprising a heavy-chain CDR1 of SEQ ID NO: 1, a heavy-chain CDR2 of SEQ ID NO: 2, a heavy-chain CDR3 of SEQ ID NO: 3, a light-chain variable region comprising a light-chain CDR1 of SEQ ID NO: 4, a light-chain CDR2 of SEQ ID NO: 5, a light-chain CDR3 of SEQ ID NO: 40.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or an antigen-Date Recue/Date Received 2021-06-29 binding fragment thereof comprises a heavy-chain variable region of SEQ ID NO: 25.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or an antigen-binding fragment thereof comprises a light-chain variable region selected from the group consisting of SEQ ID NOS: 26, 41 to 48, and 50.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid according to claim 4.

6. A cell for producing the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, which is transformed with the expression vector according to claim 5.

7. A method of producing the antibody binding to VISTA or antigen-binding fragment thereof according to any one of claims 1 to 3 comprising:
(a) culturing the cell according to claim 6; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cell.
Date Recue/Date Received 2021-06-29

8. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 as an active ingredient and a pharmaceutically acceptable carrier for preventing or treating a tumor or an autoimmune disease expressing V-domain Ig suppressor of T-cell activation (VISTA).

9. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 for administration in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody.
Date Recue/Date Received 2021-06-29